This invention pertains to the combined use of solid substrates, micro-deposition techniques, spectral imaging methods, and data processing to facilitate the concentration and separate detection of biological polymers, oligomers, and monomers including proteins, peptides, polysaccharides, glycans, nucleotides, and other analytes including smaller molecules, impurities and buffers, in a liquid mixture, using spectral analysis such as normal Raman spectroscopy, infrared spectroscopy and matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. The term “normal Raman” is specifically adopted to distinguish the spectral imaging methods that are preferred for use in the present invention from surface-enhanced Raman spectroscopy (SERS).
Raman spectroscopy is increasingly being recognized as an important analytical tool, particularly in pharmaceutical, biomedical and biological applications, as a result of Raman's high chemical “fingerprint” information content. The vibrational spectral features present in normal Raman spectra collected from many organic molecules contain molecular “finger prints” that can be used to identify, distinguish and even quantify the molecules of different structure, conformation, branching or chemical modification. Likewise, infrared (IR) spectroscopy is a useful tool in the non-destructive analysis of materials including biological and other organic analytes. U.S. Pat. No. 5,334,837 to Ikeda et al. describes micro analytical methods and associated apparatus for detecting and quantifying organic compounds with very high sensitivity using IR spectroscopy in which drops of a liquid sample are deposited on a hydrophobic layer of from about 0.1 to 25 μm thickness located on an IR reflective or refractive surface of a sample holder that generally has a hydrophilic substrate, which can be stainless steel. Reduced suppression of absorption of infrared radiation by the hydrophobic layer is said to be achieved by reducing the thickness of the hydrophobic layer to between about 8 μm and 16 μm. Depressions or pin-holes of about 200 μm diameter spaced on 5 to 10 mm centers are provided that generally protrude through the hydrophobic layer to the hydrophilic substrate to act as condensing nuclei for the sample solution. A liquid sample solution placed in contact with a depression or hole is said to ball up while drying so as to leave a coherent concentrated dried sample in and/or adjacent to the depression or hole for subsequent examination. While there is a discussion of possible co-elution of specimen liquid to a mass analyzer for simultaneous testing, there is no provision for Raman analysis, nor is there any provision for mass spectral analysis of the identical sample. There is also no discussion of any intentional separation of the liquid sample constituents in the depression or hole of the sample holder.
Advantages of Raman relative to other vibrational spectroscopies, such as mid-IR and near-IR absorption or reflectance, include the relative insensitivity of Raman to water as well as Raman's compatibility with optical microscopy and CCD camera detection methods. However, since Raman scattering has a much lower cross-section than fluorescence, normal Raman spectral features can be easily obscured by even trace quantities of fluorescent impurities. Furthermore, Raman scattering from solvents containing buffering agents can overwhelm the normal Raman scattering from biological compounds that are present at much lower concentrations. The signal integration time for Raman is also generally greater than for some other vibrational spectroscopes. Although normal Raman spectroscopy has typically been restricted to high concentration condensed phase materials, that is liquids and solids, recent studies have demonstrated methods for obtaining high quality normal Raman spectra of biological compounds derived from increasingly lower concentration solutions. For example, a Raman system with enhanced collection efficiency has reportedly obtained normal Raman spectra from protein solutions with concentrations in the 10−4 M range. A liquid core waveguide has reportedly been used to measure normal Raman spectra of proteins in the 10−5 M concentration range with a total of about 10 nmol of protein probed using a 24 mW, 532 nm, excitation laser with an integration time of 3 minutes.
Various methods have previously been used to combat fluorescence background interference, none of which are universally applicable. Since fluorescence requires optical absorption, fluorescence can in some cases be effectively suppressed by using an excitation laser of either longer or shorter wavelength than the chromophore absorption band. Alternatively, fluorescence can be suppressed using time-resolved methods with gated or lock-in detection to distinguish Raman scattering from delayed fluorescence. A less sophisticated, but often quite effective, method for reducing fluorescence is photo-bleaching of the fluorescent compounds by prolonged exposure to the Raman excitation laser. In addition, fluorescence can be quenched by conductive solid substrates such as metals or graphitic materials. However, since efficient quenching requires direct contact with the conductive solid substrate, this method is only applicable to monolayer structures. Raman spectroscopy on such monolayer structures require either very high laser powers and cryogenic cooling, or electromagnetic and/or resonance enhancement of the monolayer Raman scattering, i.e. resonance Raman (RR) techniques.
Since Raman is a scattering rather than absorption process Raman testing can be performed on either optically transparent or opaque samples and substrates. Furthermore, no optical tagging or other chemical pre-processing of the sample is required, although additional benefit can in some cases be derived from the use of Raman tags. A large number of previous studies have described the use of RR and SERS to increase the Raman scattering cross section of various compounds, including biological materials. RR enhancement requires an analyte with a chromophore that absorbs at the wavelength of excitation, which for most proteins is in the UV. Furthermore, RR only enhances Raman features that are strongly coupled to the chromophore and thus can omit other important structural information. An additional limitation of RR is the optical damage induced by heating and/or photochemistry. SERS, on the other hand, can provide even larger enhancement than RR using suitable nano-structured metal substrates. Key limitations of SERS include the generation of spurious background signals, optical damage susceptibility and often poor reproducibility, both in terms of absolute intensity and spectral shape. It is also noteworthy that most SERS studies of biological compounds have been performed using mM analyte concentrations. These comments should not be taken to imply that RR and SERS are not tremendously powerful bio-molecule analysis methods that can provide valuable structural and kinetic information and unrivaled single-molecule sensitivity.
The most powerful current methods for proteomic diagnostics are those based on mass spectroscopy, particularly MALDI TOF MS, as these may be used to determine amino acid sequence, post-translational modification and even protein-protein interactions. High detection sensitivity combined with high sample throughput can be achieved with the use of a pre-structured sample support that confines each diagnostic specimen to a spot of less than 200 μm diameter. This sample geometry is achieved with stainless steel sample supports coated with a 30 to 50 μm thick polytetrafluoroethylene (PTFE) layer onto which an array of gold islands is deposited using a photolithographic mask. The islands, formed as 200 μm diameter round disks located at 2.25 mm spacing, act as hydrophilic sample anchors that hold liquid sample droplets in place during solvent evaporation. The water-repellent PTFE surface surrounding each island ensures that the droplets remain located on the gold islands so that, after evaporation, uniform specimens of known geometry are located at predetermined positions on the support (Bruker Anchorchip). However, accurate quantification remains a significant challenge and ambiguities can exist in the analysis of biological and organic analytes of different structure but equal mass. Thus the sensitivity of normal Raman spectroscopy to differences in conformation, branching and binding can be useful to compliment mass spectroscopy by providing a valuable additional dimension of chemical structure information. Furthermore, normal Raman can in some cases lead to a stand-alone detection, identification and/or quantification method for proteomic analysis.
Thus, there remains a need for a method for separating and detecting the presence of analytes, particularly proteomic analytes, at very low concentrations in liquid specimens containing much larger concentrations of other molecules of potentially masking character. There remains a further need for non-destructive testing procedures for selected analytes that can be repeatedly performed on any given specimen, preferably using multiple techniques to accumulate a library of information on a given sample. There also remains a need for very simply eliminating fluorescence interference from the normal Raman spectra of impure solids, for example, biological amino acids and proteins. There is also a need for a sample support that can be used in IR spectroscopy, Raman spectroscopy and MALDI mass spectroscopy that exhibits low optical absorbance, high optical reflectance, little or no interfering background signals, and, a non-wetting interaction with the analyte solution, i.e., low solvent affinity, and that is useful with a range of solvents. There is a further need for methods of handling extremely small volumes of sample liquids containing analytes at very small concentration.